The bacterium Bordetella pertussis causes the disease "whooping cough". The vaccine in current use in the United States causes unpleasant side-reactions and may in rare cases cause permanent neurological damage. Reports concerning these adverse reactions threaten to decrease acceptance of the vaccine and precipitate an epidemic. The goal is to contribute to the rational design of an acceptable alternative vaccine and improve assays for the evaluation of alternative vaccines. The approach is based on evidence that: 1) some, but not all antibodies against a toxin of Bordetella pertussis, termed pertussis toxin, can confer protection; 2) that an inactive analog of the toxin should be in an alternative vaccine, and 3) the relevant activities of the toxin arise from the enzymatic ADP-ribosyltransferase activity of the toxin's S1 subunit. The specific aims are as follows: Specific Aim 1: Toxin Inactivation; A) Identify amino acids critical for the enzymatic activity of the S1 subunit. Methods used: photo-affinity labelling, anti-peptide antibodies, and characterizing putatively equivalent mutations in pertussis and cholera toxins introduced using site-directed mutagenesis. B) Describe the functional consequences of inactivating mutations in terms of binding the substrate NAD and recognition by antibodies. Specific Aim 2: Assays of Toxin Antibodies; Some but not all antibodies recognizing pertussis toxin confer protection. The goal is to develop enzymatic classification methods that identify protective antibodies. The approach involves classifying antibodies in terms of mechanisms that inhibit ADP-ribosyltransferase activity, and improving assays based on inhibition of toxin effects on cultured cells. Specific Aim 3: Immunization with Inactive Toxin Analogs; The work will test the hypothesis that, if properly administered, an inactive analog of the S1 subunit of pertussis toxin can elicit protective antibodies. The approach involves defining methods to aggregate the analog, and using pertussis toxin itself as an adjuvant.